Measuring tobacco smoke exposure: quantifying nicotine/cotinine concentration in biological samples by colorimetry, chromatography and immunoassay methods
Introduction
A large body of evidence indicates that tobacco smoking has unfavorable consequences on human health [1], [2], [3], [4], [5], [6], [7]. Chronic smokers run the risk of lung cancer [2], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], respiratory infections [18], [19], heart disease [2], [20], [21], [22], and pregnancy complications [23], [24] caused by inhalation of nicotine, the principal component of tobacco. The annual worldwide mortality due to tobacco use is estimated to be 3 million [25]. Many hazardous substances in mainstream cigarette smoke are also present in environmental tobacco smoke (ETS). Therefore individuals involuntarily exposed to ETS, called passive smokers, are also adversely affected. For example, there are about 3000 lung cancer deaths per year among nonsmokers [26]. Even infants nursed by smoking mothers are affected by nicotine as it is secreted in the milk [24], [27], [28], [29].
Often, a distinction has to be made between smokers and nonsmokers, and between non smokers exposed to ETS and non smokers not exposed to ETS. For example, life insurance companies are interested in knowing the smoking status of potential insurance customers, since heavy smokers run the risk of decreased life expectancy. In this respect, biochemical measurements with appropriate markers have been found useful [30], [31]. A threshold value of 500 ng ml−1 of cotinine, a major metabolite of nicotine, is used to distinguish smokers from nonsmokers [32].
This article reviews the procedures used to assess tobacco smoke exposure. Specifically, it compares different biomarkers used to determine the smoking status of an individual and the different methods used to extract these biomarkers from saliva, urine, and blood. Advantages and disadvantages of all the assays currently used are discussed. The article also reviews methods for evaluating the cortisol levels in tobacco smokers; cortisol is a stress hormone that appears to be closely linked to nicotine metabolism [33]. Recent review articles regarding tobacco smoke exposure include a mini-review on the use of urinary cotinine as a tobacco-smoke exposure index [34] and a review on the use of cotinine as a biomarker of environmental tobacco smoke exposure [35].
Section snippets
Nicotine metabolites
Nicotine is metabolized to more than 20 different derivatives [36]. In humans, 70% of nicotine is oxidized to cotinine, 4% is oxidized differently, 9% is excreted unchanged in the urine, and the metabolic outcome of the remaining 17% is still unknown [37], [38], [39], [40]. (Scheme 1).
Tobacco smoking also produces metabolites other than those derived from nicotine, such as trans, trans-muconic acid [41], [42] and 1-hydroxy pyrene [43]. These metabolites are produced from benzene [41], [42] and
Biomarkers for assessing smoking status
An ideal marker for assessing the smoking status of individuals should have a reasonable half-life, be specific, be amenable to estimation in body fluids, and be available at concentrations that can be quantified using existing analytical methods. The presence of other compounds should not interfere in the estimation of the marker and the marker should not be influenced by environmental sources other than tobacco smoke. The markers used for assessing the smoking status of individuals, and the
Collection of body fluids
The receptacle for collecting body fluids should be made of either glass or polypropylene, should be disposable, should be kept in a sealed package protected from the environment, and precleaned and siliconized before use [6]. A polypropylene receptacle with a screw cap closure is preferred since it can withstand breakage during transportation to a distant laboratory [65]. Different biological fluids are collected as follows.
Methods for the extraction of biomarkers
Several organic solvents have been employed to extract organic constituents in plasma, saliva, and urine. From the organic solvent the basic constituents, namely, nicotine and cotinine are recovered by salt formation with a mineral acid (HCl/H2SO4/H3PO4) [73]. Cotinine and/or nicotine can be re-extracted from the corresponding salts by basification by NaOH (or Na2CO3–NaHCO3 [79], [80], [81], [82]/K2CO3–NH4OH [74]). These are then assayed either by GLC [79], [83], [84], HPLC [39], [65], GC–MS
Colorimetry
For monitoring the smoking status of humans, colorimetry is a desirable method of analysis. It is simple, inexpensive, and gives (after taking appropriate precautions) [87], [88] an estimate of total metabolites produced from nicotine inhaled during smoking [77], [89], [90]. It, however, lacks specificity and the estimated urinary concentration is reported to be higher than that obtained by gas or liquid chromatography. Moreover, drugs containing the pyridine nucleus (e.g., isoniazid,
Cortisol
Cortisol is one of the most frequently used steroid hormones for assessing adrenal disorders. Serum and urinary cortisol concentrations have been used to monitor adrenocortical function as well as in the diagnosis of chronic fatigue and depression [152], [153]. Cortisol is also used as a biomarker of stress [154], [155], [156], [157]. A study performed on rats concluded that stress lowers circulating nicotine levels [33]. In humans, the relationship between stress and smoking is well documented
Conclusion
Of the different body fluids, saliva is the matrix of choice for assessing the presence of nicotine and its metabolites in humans exposed to ETS. Of the different biomarkers, cotinine appears to be the analyte of choice, as it fulfills the prerequisites of specificity and retention time and is found in detectable concentrations in all three matrices. GC–MS is preferred for analyzing nicotine and its metabolites in smokers whereas GLC is preferred for studies related to passive smokers.
Acknowledgements
The author thanks Christina Quadraccio and Steven Gilday for assisting in the literature search.
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